Currently, therapeutic interventions for stroke are limited and those that are effective are limited to administration relatively soon after the insult. New therapeutic interventions are needed that can be administered in the hours and days following a stroke that improve long-term outcome. Repetitive waves of spreading depression (SD) have been argued to contribute to ischemic infarct development in animal stroke models and recently strong evidence has shown that these repetitive spreading events occur in human stroke patients for a period of days following the onset of an ischemic insult. Each SD imposes a massive metabolic challenge on neurons and astrocytes as ionic balances are greatly disrupted. When combined with metabolic compromise in peri-infarct tissue, the additional severe metabolic load of SD is likely to contribute to tissue death. Finding ways to limit the onset or the consequences of SDs in the ischemic brain has therefore become a major goal of current research. Using genetic mice lacking Zn2+ in synaptic vesicles, I have shown a large amount of synaptic Zn2+ release with each SD event generated in hippocampal slices. Zn2+ can accumulate in postsynaptic cells following presynaptic release and it is known that excessive accumulation of Zn2+ is highly toxic to neurons and glia. These observations lead to my overall hypothesis that synaptic Zn2+ release during SD contributes to neuronal injury in ischemia models. Experiments in Aim 1 will characterize the synaptic release of Zn2+ in neocortical tissues using acutely prepared slices from wild-type mice or mice lacking vesicular Zn2+. Additional studies will be performed to investigate the consequences of Zn2+ released by SD in an in vitro ischemia model. Studies in Aim 2 will utilize a potent vasoconstrictor, endothelin-1 (ET-1) to induce an in vivo focal ischemic lesion shown to induce repetitive SDs. Using this model, I will determine whether synaptic Zn2+ release is significant during SD in vivo, and determine the consequences of synaptic Zn2+ release on ischemic lesion volume in this model. Zn2+ is known to be neurotoxic in the post-ischemic period;however, the source of Zn2+ is not. This proposal aims to investigate where the Zn2+ is originating and sheds new light on the contributions of synaptic Zn2+ release during SD in the post-ischemic period. The added training and experience that this project will give me will help me to obtain my overall career goal of becoming an independent investigator and make significant contributions to the stroke and neurodegenerative fields. PUBLIC HEALTH RELEVANCE: Following a stroke, waves of inhibition of brain activity spread from the initial site of injury and contributes to the enlargement of the ischemic injury. The research in this proposal is designed to investigate mechanisms by which these events lead to tissue death. If successful, these findings should contribute to the development of novel therapeutic interventions that can be applied to a much larger population of stroke patients than is possible with current treatments.